The protein kinase which catalyzes the phosphorylation of the hydroxyl group positioned in the tyrosine and serine/threonine residue of a protein manages the role of being important to the proliferation, differentiation, survival, migration, and invasion of a cell. The protein kinase is one of the most important commanders for controlling the signal transduction system within an organism. The signal transduction system in the living cell should be properly and smoothly turned on and put out for the maintenance of homeostasis of an organism. But the collapsed signal transduction system caused by the mutation or the overexpression of the specific protein kinases induces the various disease such as a cancer, an inflammation, a metabolic disease, a brain disorder and so forth. 518 kinds of human protein kinases have been identified or proposed so far, which is corresponding to about 1.7% of the human total gene exist. It bisects in the tyrosine protein kinase (over 90 kinds) and serine/threonine protein kinase. The tyrosine protein kinase can be classified into 20 subfamilies composed of 58 kinds which are receptor tyrosine kinases and 10 subfamilies composed of 32 kinds that belong to cytoplasm/non-receptor tyrosine kinases (Manning et al., Science, 2002, 298, 1912). The extracellular domain of the receptor tyrosine kinases can accommodate their ligands such as the growth factor and the kinase domain of them located in a cytoplasm can phosphorylate specific tyrosines. Once a ligand is binding to extracellular domain of a receptor tyrosine kinase, the receptor tyrosine kinase is dimerized and specific tyrosines in the cytoplasm domain become autophosphorylated. In turn, a signaling is progressed through the successive phosphorylation of downstream proteins in the cytoplasm and nucleus and this successive signaling eventually render transcription factors which cause cancer become activated and overexpressed. The Raf which belongs to serine/threonine (Ser/Thr) protein kinase family is responsible for transducing signal generated from activated growth factor receptor in cell membrane to the nucleus. The first identified rafgene is oncogenic v-raf (Mark G E et al., 1984). The signal transduction system derived from mitogen activated protein kinase (MAPK) is essential for cell proliferation, division, survival, death and invasion as well as transcription regulation and development. The MAPK signaling system works mainly by the sequential phosphorylation process of MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK. The Raf, MEK, and ERK (extracellular signal-regulated kinase) are corresponding to MAPK kinase kinase (MAPKKK, MAP3K), MAPK kinase (MAPKK, MAP2K) and MAPK respectively.
The Ras, a small GTP binding protein activated by signal from growth factor receptor render Raf-MEK-ERK sequentially phosphorylated and ultimately sequential MAPK signaling is transduced to the nucleus. Raf family kinase consists of A-raf, B-raf, and c-raf (raf-1). Raf-1 has most extensively been studied up on these three Raf isoforms. Ras tumor genes (especially k-Ras) which are constitutively active turned out to be implicated with pancreatic cancer (approximately 90%), rectal cancer (approximately 45%), liver cancer (approximately 30%), three non small cell lung cancer (approximately 35%), kidney cancer (approximately 10%) and other various solid tumors. The Raf-1 in conjunction with activated Ras could be activated once serine 338 of Raf-1 is phosphorylated (Avruch, J. et al., Recent Progress in Hormone Research, 2001, 56, 127). In contrast, the 14-3-3 protein combined with Raf-1 of which serin 259 is phosphorylated makes Raf-1 inactivated. Raf is also involved in NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathway and plays an important role in the immune response and inflammation (Caraglia, M. et al., Annals of Oncology, 2006, 17, 124). Raf phosphorylates inactive IKBs (Inhibitor of KB) protein and induces NFkB to be located in the nucleus and ultimately up-regulates transcription factors which inhibit cell death.
The dissimilar mechanism for the antiapoptotic of Raf is as follows. Raf binds to Bcl-2 to form Raf-Bcl-2 dimer and does the location shift to a mitochondria. If it makes Bad protein phosphorylated in that place, then the antiapoptotic function of Bcl-2 operates. The Raf is immunoprecipitated with Bcl-2-(Yuryev, A. et al., Mol. Cell. Biol. 2000, 20, 4870). Three subtypes (A-Raf, B-Raf, C-Raf/Raf-1) of the Raf protein bear the N-terminal control domain and preserved three domains (CR1, CR2, CR3) in the C-terminus kinase domain. CR1 includes the Ras binding domain in which a cystein is abundant. And CR2 has binding site for 14-3-3 protein i.e. a serine 259 of Raf-1. CR3 contains the catalytic domain (Tran et al., J Biol Chem, 2005, 280, 16244) and owns auto-phosphorylation site to be fully activated. For example, the phosphorylation of the threonine 491 and serine 494 of Raf-1 (Wellbrock, C. Nature Reviews Molecular Cell Biology, 2004, 5, 875) enables Raf-1 to be maximally activated.
Three subtypes of the Raf protein are overexpressed in different tissues. C-Raf is ubiquitously expressed in nearly all tissues, on the other hands, A-Raf is mainly expressed in urogenital apparatus (kidney, uterus and the prostate gland) and B-Raf is mainly found in a nerve, a spleen, and the blood forming organ (Jaiswal, R. K. et al, J. Biol. Chem., 1966, 271, 23626) (B-Raf (SEQ. ID NO. 1)).
B-Raf mutation is linked with about 7% of all of the human cancer. Especially, the mutation of B-Raf is observed in the frequency in which it is high in the melanoma (about 70%) that is a kind of the skin cancer. The B-raf-V600E mutant species in which the valine 600 positioned in Exon 15 is replaced by glutamic acid due to point mutation is mainly (about 90%) found in melanoma (Davies, H. et al, Nature 2002, 417, 949) (B-raf-V600E (SEQ. ID. NO. 2)). In vitro kinase activity of B-raf-V600E is about 500 times higher than that of wild type B-Raf. Accordingly, B-raf-V600E makes MAPK kinase signal transduction system be overactivated and induces various types of cancers. The reason why the kinase activity of B-raf-V600E is higher compared with wild type B-raf is as follows. The point-mutated glutamic acid 600 positioned in the B-Raf activation segment acts like phosphate group between phosphorylation sites (threonine 598 through serine 601). This phosphate mimicking induces conformation change which lead B-Raf kinase domain to be constitutively activated (Tuveson, D. A., Cancer Cell, 2003, 4, 95). The meantime till now identified B-raf mutant species are about 40. These mutations are significantly occurred in the activation segment and G-loop in which a glycine is abundant. The generation frequency of other mutant species except V600E is remarkably low. In the colorectal cancer, about 10% of the B-Raf mutant species are generated in the G-loop of the kinase domain (Rajagopalan et al., Nature 2002 418, 934).
The autologous suppression (auto-inhibition) domain exists in the N-terminal of B-Raf. However, B-Raf becomes always active once the activated H-Ras binds to B-Raf. This is formed through the phosphorylation of the serine-445, which corresponds to the phosphorylation of C-Raf serine-338. The B-Raf V600E mutant species interrupts the autologous suppression of B-Raf and renders B-Raf to beconstitutively active.
Moreover, the B-Raf-V600E mutant species is detected at the frequency in which it is high in the papillary thyroid cancer (about 50%) (Salvatore, G. J. Clin. Endocrinol. Metab. 2004, 89, 5175). The B-Raf-V600E mutant species is also implicated with the colon carcinoma (about 20%) and uterine cancer (about 30%). Meantime, the overactivivty of C-Raf is observed in the kidney cancer (renal cell carcinoma) and liver cancer (HCC) in about 50% and almost 100% frequency respectively without the expression of the mutant species.
Sorafenib (the BAY 43-9006/the trademark Nexavar) developed by Bayer and Onyx company strongly suppresses C-Raf, wild typeB-Raf, or the mutant species B-Raf. Moreover, sorafenib impedes the kinase activity of several receptor tyrosine kinases including the platelet-derived growth factor receptor, the vascular endothelial growth factor (vascular endothelial growth factor receptor 1/2/3), the fibroblast growth factor receptor, Flt-3, c-Kit, RET, and so forth. Sorafenib suppresses kinase activity through the mechanism in which it stabilizes DGF motif of the kinase domain to have the deactivated conformation (inactive conformation) (Wan, P. T. et. al. Cell, 2004, 116, 855). Sorafenib gained the approval from FDA as a therapeutic agent for advanced renal cell carcinoma in 2005. However, the clinical benefits of Sorafenib treatment is caused by the complex suppression of several kinases including the vascular endothelial growth factor (vascular endothelial growth factor receptor 1/2/3) than soley Raf impediment. In the phase II clinical investigation, The MTD (maximum tolerated dose) of Sorafenib was 400 mg twice a day. The administration of 600 mg Sorafenib twice a dayinduces the skin toxicity side effect of the grade 3. The skin of hand and foot gets stripped off and the common side effect of Sorafenib is the erythema, and hand-foot syndrome of the edema symptom. Meantime, sorafenib received the approval as the hepatocellular carcinoma (HCC) therapeutic agent in 2008. Moreover, in the phase II clinical trial, Sorafenib demonstrated reasonable efficacy against the thyroid cancer, metastatic prostate cancer, and breast cancer. In the meanwhile, Sorafenib revealed a mild therapeutic efficacy against the melanoma that is a kind of skin cancer. Meantime, PLX4-720, 7-azaindole derivative developed by Plexxikon induces the apoptosis of the melanoma cell line such as 1205Lu (raf-V660E overexpression cell strain) (Tsai, J. et. al. PNAS, 2008, 105, 3041). PLX-4720 strongly impedes the kinase activity of Raf-V660E with an IC50 value of 13 nM and exhibits good anti-proliferative activity (IC50=0.5 μM) against the A375 melanoma cell line (raf-V660E overexpression cell strain). CHIR-265 developed by the Norvatis/Chiron strongly inhibits several kinases like B-Raf-V600E (IC50=19 nM), KDR (IC50=70 nM), PDGFR-b (IC50=30 nM), and c-Kit (IC50=20 nM). CHIR-265 is under phase I clinical investigation for melanoma treatment. Recently, the resistance problem of the Raf inhibitors comes to the front. Montagut and collegues did culture of M14 cell strain (human body melanoma cell line) that bears the B-Raf-V600E mutant species in the presence of Raf inhibitor (AZ628) and obtained the clones which resist against Raf inhibitor (AZ628). They explained the mechanism for resistance occurence of Raf inhibitor on the basis of this experiment. If B-Raf is inhibited, the protein expression level of C-Raf increases and the drug inhibitory activity against B-Raf-V600E falls down. But the susceptibility of the geldanamycin, HSP90 inhibitor increases on the melanoma cell line which resists against the Raf inhibitor (AZ628). Therefore, HSP90 inhibition can override the resistance problem of the Raf inhibitor (Montagut, C. Cancer Research, 2008, 68, 4853).
VEGFR-2 (Vascular endothelial growth factorreceptor-2) also referred to as KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase) belongs to the class III family of receptor tyrosine kinases and is deeply implicated with angiogenesis process that ultimately leads to new blood vessels formation from pre-existing vessels. Angiogenesis is associated with pathological conditions such as cancer, rheumatoid arthritis, diabetic retinopathy, and neovascular glaucoma. It has widely been known that VEGFR-2 inhibition suppress angiogenesis and VEGFR-2 has been regarded as an attractive molecular target for cancer therapeutics.
VEGF is mainly generated in the blood vessel endothelial cell, the hemopoietic cell, and the stromal cell in the hypoxic state and by the stimulation of TGF, interleukin, and cell growth factors like PDGF. VEGF binds to the VEGF receptor (VEGFR)-1, -2, and -3. The signal specificity of VEGFR is more delicately controlled by a neurophilin, the heparan sulfate, the coreceptor (assisted receptor) including cadherin and integrin.
The biological function of VEGF is mediated through the type III RTK, the VEGFR-1 (Flt-1), the VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4). VEGFR becomes closely related to the Fms, Kit, and PDGFR. Whereas VEGF-A unite combines with VEGFR-1 and -2, VEGF-C binds to VEGF-2 and -3. Whereas VEGF-A and -B preferentially need for the angiogenesis, VEGF-C and -D are essential to the lymphangiogenesis. The new blood vessels make pass role for supplying nutrient and oxygen to a tumor and play a role for the cancer cell metastasis, which are essential to proliferation and invasion of cancer cell. In case of normal cell, the angiogenesis is made to be balanced within an organism by the mutual regulation of the angiogenesis accelerated substance (angiogenic stimulator) and suppression of angiogenesis material (angiogenic suppressor) but in case of cancer cell, this balance is broken and VEGFR is activated by VEGF (vascular endothelial growth factor) which most reaches the big effect to the vascular endothelial cell. The small molecule inhibitors which suppress the receptor tyrosine kinases such as VEGFR have been variously studied and developed as there is high possibility that these anti-angiogenicagents can be used for treating most of the solid tumors. In addition, they have the advantage of expecting therapeutic efficacy with relatively lower side effect compared with cyto-toxic anti-cancer agents.
A lot of small molecule VEGFR-2 inhibitors have been identified and pursued and some of these are currently under clinical investigation (Schenone et al., Curr. Med. Chem. 2007; 14:2495.). Sorafenib and Sunitinb that are multi-targeted tyrosine kinase inhibitors including VEGFR-2 inhibition have already been launched.
Another receptor tyrosine kinase related to angiogenesis is Tie-2 expressed predominantly on vascular endothelium. Tie-2 has also been found in haematopoietic cells. Angiopoietins, Ang1 and Ang 2 were discovered as Tie-2 ligand (Davis et al., Cell 1996; 87:1161.). Ang 1 binding to extracellular domain of Tie-2 leads to auto-phosphorylation of Tie-2 kinase domain but curiously does not activate MAPK nor does it stimulate mitogenesis. It seems likely that Ang 2 plays crucial functions in the lymphatic vascular system. It has already been proved Tie-2 inhibition with dominant-negative Tie-2 receptor reduces tumor angiogenesis and growth in mice (Lin et al., Proc. Natl. Acad. Sci. USA 1998; 95:8829.).
The RET (rearranged during transfection) protooncogen primarily expressed in the nervous and excretory systems belongs to receptor tyrosine kinase family. N-terminal extracellular domain of RET is composed of four cadherin-like repeats, a calcium-binding site, nine N-glycosylation sites, and a cysteine-rich region (Airaksinen et al., Nat. Rev. Neurosci. 2002; 3:383.). At least 12 tyrosine autophosphorylation sites are located in the cytoplasmic domain of RET isoforms (Liu, J. Biol. Chem. 1996; 271:5309.). For instance, there are 16 tyrosines in the intracellular kinase domain of RET9 isoform. GFUGFR-α complex binding to extracellular portion of RET renders kinase domain of RET autophosphorylated and activated (Aiaksinen et al., Nat. Rev. Neurosci. 2002; 3:383.). GNDF ((glial-derived neurotropic factor) family ligands, GFL is composed of GDNF, artemin, neurturin, and persephin. GNDF family receptor-, GFR-α composed of four subtypes (GFR-α 1-4) represents glycosylphosphatidylinositol-anchored coreceptors. It has been reported that RET plays important roles in the development of parasympathetic and enteric nervous systems and kidney in mice (Pachnis et al., Development1993; 119:1005.). RET lose of function caused by germline mutation is implicated with Hirschsprung's disease (Manie et al., Trends Genet. 2001; 17:580.) that is defined as congenital aganglionosis of the distal intestines. In contrast, gain of function RET mutation is found to be associated with human cancers such as multiple endocrine neoplasia type 2A (MEN2A), MEN2B, and familial medullary thyroid carcinoma (FMTC). In particular, RET turns out to be a promising molecular target of thyroid cancer therapeutics (Cote and Gagel, N. Engl. J. Med. 2003; 349:1566.). Bcr-Abl that is a oncogenic fused tyrosine kinase is generated by abberant translocation of Bcr (breakpoint cluster region) gene located on chromosome 22 and Abl (V-abl Abelson murine leukemia viral oncogene homolog) gene from chromosome 9. This characteristic chromosomal translocation is referred to as Philadelphia chromosome (Nowell and Hungerford, J. Natl. Cancer Inst. 1960; 25:85.). The size of Bcr-Abl fused protein is determined by the breakpoint of Bcr compartment. Out of three isoform (190, 210, 230 kDa) of Bcr-Abl implicated with leukemia, p210Bcr-Abl has proved to be sufficient for causing CML (chronic myeloid leukemia). Gleevec (Imatinib mesylate) of which chemotype is phenylaminopyrimidine (PAP) has opened new era in drug discovery field as Gleevec is the first drug for molecularly targeted therapeutics. Unfortunately, Gleevec recently suffers from acquired drug resistance mainly due to several kinds of point mutations that have emerged in the kinase domain of Abl. Next generation of Gleevec is significantly required to override Gleevec resistance by inhibiting all of clinically relevant point mutants including T3151-Bcr-Abl even though the second generation of Gleevec such as Nilotinib and Dasatinib has already launched on the market. Nilotinib and Dasatinib are unfortunately inactive on including T3151-Bcr-Abl that turned out to be clinically the most serious point mutant.